When human immunodeficiency virus (HIV) infection is controlled by highly active antiretroviral therapy (HAART), HIV persists in a reservoir of immune cells that are difficult to distinguish from comparable uninfected cells. A key obstacle to eliminating this reservoir is identifying what makes these cells unique. We hypothesize that the developmental history of infected CD4+ T cells is perturbed in ways that can be measured even while HIV is latent. However, we are currently unable to specifically study the reservoir cell states because to determine if virus is present one must activate and alter the cell. To identify these cells an ideal experimental model system would activate an irreversible genetic switch in all HIV infected cells to mark all cells that have survived infection. The use of humanized mice in research provides an opportunity to genetically manipulate human cells that develop and become infected with HIV in an in vivo context. Immunodeficient mice that are xenografted human immune systems can be infected with HIV and display a progressive decline in CD4 T cells that can be treated with highly active antiretroviral therapy. When treatment is withdrawn plasma viremia rebounds. Here we propose to develop a method to engineer humanized mice engrafted with a human immune system that encodes an irreversible genetic switch to mark latent viral reservoirs. Using state of the art lentiviral transduction we will directly transduce hematopoietic stem cells with the HIV sensor construct. A potential shortcoming of this approach is that this method does not result in the uniform modification of all human cells. In preliminary studies, we describe a major breakthrough in stem cell reprogramming whereby human fibroblasts can directly induced to become human hematopoietic stem cells to generate a multilineage reconstitution of immune cells in when transplanted into NOD/SCID/IL2Rgc-/-(NSG) mice. We will combine this novel approach with recombinant tools that encode sensitive and specific HIV activated switches that are developed in clonal fibroblast lines, and these will be reprogrammed into induced hematopoietic stem cells (iHSC) to create novel humanized mice. The goal of this technological proposal is to develop a genetic reporter mouse model where all infected cells express a GFP lineage marker to physically isolate the reservoir for molecular characterization. We will examine the inducibility of the provirus, the surface phenotype genetically marked cells that have survived HIV infection. With a fluorescent tag on the infected cells, it then becomes possible to obtain the transcriptional profile of these cells t better understand the cellular state that maintains viral persistence. Using this unbiased approach, we hope to discover specific genes and/or cell states that would allow us to target or activate this latent reservoir.